Die Prostata-Zelllinien werden freundlicherweise von Dr. Ling-Yu Wang und Dr. Hsing-Jien Kung bei Cancer Center, UC Davis zur Verfügung gestellt. Dieses Projekt wird von NIH Galvanotaxis Zuschuss 4R33AI080604 unterstützt.

1. Die Kultivierung Prostatazellen <ol><li> Kultur die PRNs-1-1 und PNT2 Prostata-Zellen auf 100 mm Kulturschalen in RPMI 1640 Medium mit 10% FBS und Antibiotika-Antimykotische ergänzt bei 37 ° C mit 5% CO 2. Aktualisieren Sie das Kulturmedium täglich, bis die Zellen bis zu 80% Konfluenz für die Galvanotaxis Experimente. </li></ol> 2. Montage Galvanotaxis Chambers <ol><li> Montage Unterkammern <ol><li> Wischen eines Kunststoff Galvanotaxis Kammer mit 2-Propanol. Übernehmen Sie die Meeressilizium Versiegelung rund um die kreisförmigen Öffnungen an der Unterseite einer Kammer mit einer Spritze und legen einen großen Deckglas (Abbildung 1). Verwenden Sie die Rückseite eines Baumwoll-Applikator, um das Deckglas nach unten drücken, und wischen Sie das überschüssige Kleber mit Wattestäbchen. Die kreisförmigen Öffnungen kann das Medium und elektrischer Strom über die Zellen zwischen den beiden inneren Medienspeicher zu fließen. </li><li> Schneiden Sie ein kleines Deckglas 6 mm x 2 machen5 mm Abstandshalter mit einer Diamantspitze Marker. </li><li> Drehen Sie die Kammer. Auftragen von 2 Streifen aus dem Silikonkleber mit einer Spritze und / oder einem Metallspatel um eine 10 mm x 25 mm Kanaloberfläche für die Zellanheftung zwischen den kreisförmigen Öffnungen 2 an der unteren Glas erstellen. Kleben Sie zwei Stücke aus Glas Abstandhalter zwischen den zwei kreisförmigen Öffnungen. Benutzen Sie die Rückseite der Watte sich die Abstandshalter zu schieben und wischen Sie das überschüssige Kleber mit Wattestäbchen und / oder Zahnstocher. </li><li> Trocknen Sie die Kammer für 24 Stunden, bis die Silikonkleber ausgehärtet wird. Weichen Sie die Kammer mit destilliertem Wasser über Nacht, um die Essigsäure Rückstand aus der Kleber zu entfernen. Trocknen Sie die Kammer für den sofortigen Einsatz, oder die Kammer für eine spätere Verwendung gespeichert werden in einem sauberen Behälter. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> Abbildung 1: Montage des unteren Galvanotaxis Kammer. A) Eine große Deckglas wird auf den Boden eines Reinraums. B beigefügt) TWO Glasstücke Abstandshalter werden zwischen den kreisförmigen Öffnungen 2 verklebt, um eine 10 mm x 25 mm Kanal für Zellen zu befestigen erstellen. </li></ol></li><li> Seeding Zellen auf den Galvanotaxis Kammern <ol><li> Bereiten Sie die Anzahl der benötigen Galvanotaxis Kammern für das Experiment benötigt. Jede Zelllinie oder Behandlung erfordert einen einzelnen Galvanotaxis Kammer. Wischen Sie die Galvanotaxis Kammern mit 2-Propanol. Waschen Sie die Kammern mit sterilem PBS 3 mal und überprüfen Sie den Flüssigkeitsstrom zwischen den zwei kreisförmigen Öffnungen in den Kammern. </li><li> Umschließen die Kammern in sterile Zellkulturschalen, und es ihnen ermöglichen, bei 37 ° C für 15 Minuten äquilibrieren. Trennen der mit Prostatazellen aus ihrem Kulturschale mit 5 ml 0,25% Trypsin-EDTA bei 37 ° C für 5 min. Neutralisieren die Trypsin-EDTA mit 5 ml 10% FBS in PBS. </li><li> Übertragen der Zellen auf 15 ml-Röhrchen und Zentrifugieren der Zellen 5 min bei 200 × g, 37 ° C. Saugt den Überstand und resuspendiere die Zellen in 1 ml Kulturmedium. </li><li> Nehmen Sie 20 μl der Zell-Lösung auf eine Zählkammer laden und berechnen Sie die Anzahl der Zellen. Stellen Sie die Zellkonzentration bis 8 × 10 4 Zellen / ml mit Kulturmedium. </li><li> Nehmen Sie die Galvanotaxis Kammern aus dem 37 ° C-Inkubator. Seed 350 ul der Zellsuspension auf jeder Kammer. </li><li> Die Zellen in der Kulturschale mit nassen Kimwipe oder in einer feuchten Kammer in den Inkubator bei 37 ° C mit 5% CO 2 inkubieren in den Kammern über Nacht. </li></ol></li></ol><img alt="Abbildung 2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> Abb. 2: Aussaat der Zellen in die Kammer der unteren Kammer wird getrocknet und gereinigt A) Prostata-Zellen trypsiniert, gezählt und in die Kammer überführt und über Nacht inkubiert B) Ein Top Deckglas angebracht ist, um die Kammer bevor die Dreharbeiten zu versiegeln... <ol start="3"><li> Assembling die oberen Kammern <ol><li> Montieren Sie die obere Kammer vor den Dreharbeiten. Das Mikroskop ist nur Platz Abbilden eines einzigen Galvanotaxis Kammer zu einer Zeit. Warm up 10 ml Kulturmedium bei 37 ° C für jeden Galvanotaxis Kammer. Übertragen einer Galvanotaxis Kammer aus den Brutkästen zu einer 37 ° C Warmhalteplatte. </li><li> Spülen Sie die Zellen mit Kulturmedium, um die ungebundene Zellen zu entfernen. Leave 400 ul Medium in der Kammer. Mit einer Spritze Hochvakuumfett auf der Oberseite der beiden Glasabstandshalter anwenden. </li><li> Fügen Sie eine kleine Deckglas, um die Kammer zu versiegeln. Drücken Sie vorsichtig das Deckglas mit der Rückseite eines Baumwoll-Applikator. Wischen Sie das überschüssige Medium mit Wattestäbchen. </li><li> Trocknen Sie die Glasoberfläche und gelten Hochvakuumfett, um die Lücke zwischen dem Deckglas und der Kammer zu versiegeln. Verwenden Sie eine Metallspachtel, um das Fett zu verbreiten. 4 ml des Kulturmediums zu den inneren Behälter und überprüfen Sie den Flüssigkeitsstrom zwischen den Stauseen. </li></ol></li><li> Stellen AgarBrücken <ol><li> Schneiden Sie ein Paar 2-Zoll-langen PVC-Schlauch (ID 503/16 x 16.5 mm x 16.1 Wand), Flip und fügen Sie sie in einen 100-ml-Becher. </li><li> Messung 200 mg Bacto-Agar und fügen in einem 50 ml-Kolben mit 10 ml Kulturmedium zu 2% Agargel machen. Mikrowelle für 30 Sekunden. Laden Sie die Agar-Gel auf den Kunststoffschlauch mit einer Transferpipette. Lassen Sie die Agar-Brücken bei Raumtemperatur für 10 Minuten, um den Agar verfestigt. </li><li> 2 ml Kulturmedium zu den Außenbehälter des Galvanotaxis Kammer. Legen Sie die Agar-Brücken zu den inneren und äußeren Medienspeicher, um den Strom zu leiten. </li></ol></li></ol><img alt="Figur 3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> Fig. 3: Dreh die Zellen an der Kammer ein Diagramm, um die Endmontage des Galvanotaxis Kammer demonstrieren. Die Kammer ist komplett mit Hochvakuumfett abgedichtet. Medium hinzugefügt to füllen die Stauseen und zwei Agar Brücken in die Kammer eingeführt wird. Dann wird die Galvanotaxis Kammer auf den Mikroskoptisch übertragen, und die Elektroden angebracht sind, um das elektrische Feld anzulegen. Seitenansicht des zusammengebauten Galvanotaxis Kammer gezeigt, um zu zeigen, dass der elektrische Strom in den Zellen durch die Agar-Brücken und der Raum zwischen dem Deckglas. 3. Zeitraffer-Imaging <ol><li> Einschalten der Klimakammer auf 37 ° C mit 5% CO 2 in der zirkulierenden Luft 2 h vor der Bilderzeugung. </li><li> Schalten Sie das Mikroskop und starten Sie die Bilderfassungssoftware. Kalibrieren Sie den Mikroskoptisch und wählen Sie die 10X-Objektiv. </li><li> Übertragen Sie die Galvanotaxis Kammern mit dem Mikroskoptisch, sichern Sie die Kammer mit Klebeband und konzentrieren sich auf den Zellen. Setzen Sie die Galvanotaxis Elektroden an den Außenbehälter mit Kathode auf der rechten Seite. Sichern Sie die Kabel und die Elektroden mit Klebeband. </li><li> Schalten Sie die Strom box, ein elektrisches Feld an die Kammer anzuwenden. Die Ausgangsspannung mit dem Spannungsmesser über die Kammer auf 2,5 Volt für die 25 mm lange Kammer (100 mV / mm) erreichen. Pflegen Sie die Feldstärke während des Experiments durch Anpassung des Ausgangsstroms. </li><li> Wählen Sie 10 Punkte über die Kammer in der Software zu filmen zu zehn Zeitraffer-Filme erstellen. Richten Sie die Aufnahmebedingungen im 10-Minuten-Intervallen für 2 Stunden. </li><li> Starten Dreharbeiten und stellen Sie den Ausgangsstrom je nach Bedarf. </li><li> Am Ende des Experiments, entfernen Sie die Kammer von der Bühne aus und fixieren Sie die Zellen mit 95% Alkohol. Brechen Sie den offenen Kammer mit einer Rasierklinge zu reinigen und wieder verwenden. </li></ol> 4. Quantifizierung Galvanotaxis <ol><li> Drehen Sie die Zeitraffer-Filme und neu orientieren die Kathode an die Spitze der Bilder. Exportieren Sie die Filme auf die Zelle-Tracking-Software. </li><li> Manuell verfolgen die (x, y) Position 10-20 Zellen bei jedem Zeitpunkt von jedem Film (Abbildung 4 A, B).Die Migrationsabstände und die Winkel in Bezug auf die Nord-Süd-Richtung, die gleiche Ausrichtung des elektrischen Feldes in der Zelle Verfolgungssoftware (4C) berechnet werden. HINWEIS: Die durchschnittliche Wanderungsgeschwindigkeit wird von der Gesamtlaufstrecke und Gesamtdreharbeiten Zeit konvertiert. Die Laufrichtung wird von den quer zu dem Kosinus Wert umgewandelt. Wenn Zellen wandern mit zufälliger Ausrichtung, wird der Durchschnitt der Umlauf Cosinus nahe Null sein. Wenn die Zellen wandern direkt zu der Kathode, wird die Kosinuswert +1 sein. Wenn die Zellen wandern direkt zu der Anode wird der Cosinus-Wert -1 ist. </li></ol><img alt="4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> Abbildung 4: Zellverfolgung zu Direktionalität messen A) Überlagerung der Spurlinien mit Zellbildern.. Die (x, y) -Positionen der Zellen manually im Zeitraffer-Filmen verfolgt. . Wenn die Zellen wandern zufällig ist die mittlere Kosinus nahe Null B) Wenn jedoch die Zellen wandern in Richtung der Kathode oder der Anode, ist der Wert des durchschnittlichen Cosinus nahe + (Kathode) - oder. (Anode) 1.0 C ) Die Laufrichtung wird durch den Kosinus-Wert, der aus den Migrationswinkel (θ) umgewandelt wird, dargestellt. Der Kosinus (θ) gleich dem Verhältnis des Abstandes a (die Migrationsdistanz) auf Abstand b (der Abstand zu der Richtung des elektrischen Feldes projiziert). <ol start="3"><li> Speichern Sie die Messungen. Importieren der Daten in eine Datenbankanwendung, die kombinierten Ergebnisse zu berechnen. </li><li> Exportieren Sie die kombinierten Daten der durchschnittlichen Wanderungsgeschwindigkeit und Durchschnitts Kosinuswert in eine Tabellenkalkulation, um die Balkendiagramme (Abbildung 5) zu zeichnen. </li></ol>

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R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imposition+of+a+physiologic+DC+electric+field+alters+the+migratory+response+of+human+keratinocytes+on+extracellular+matrix+molecules.">Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.</a> J Invest Dermatol. 106 (4), 642-646 (1996).</li><li>Feng, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+migration+of+neural+stem+cells+derived+from+human+embryonic+stem+cells+by+an+electric+field.">Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.</a> Stem Cells. 30 (2), 349-355 (2012).</li><li>Mukerjee, E. V., Isseroff, R. R., Nuccitelli, R., Collins, S. D., Smith, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

The authors have nothing to disclose.

Die Analyse der Galvanotaxis Reaktion einer Zelle ist ein wichtiger Funktionsanzeige für viele zelluläre Migrations oder Wachstumsprozesse 27, 28. Hier nutzen wir eine maßgeschneiderte Kammer mit Glasoberfläche, um zwei Prostata-Zelllinien zu filmen. Diese Zellinien zeigten unterschiedliche Grade der Galvanotaxis, und wir vermuten, dass die intrazelluläre Lokalisierung oder die Aktivierung der Galvanotaxis vermittelnde Proteine ​​könnte während des Prozesses der Erzeugung der unsterblichen Zelllini...

Endogene elektrische Felder in verschiedenen Geweben, wie der Haut 1, 32, 33 und Gehirn 2 detektiert. Die physiologischen elektrischen Feldes dient spezifische biologische Funktionen, einschließlich Leiten Embryonalentwicklung 3, 4, Führen des Auswachsen von neuronalen Prozessen 5, 6 und Förderung Epithel- und Hornhautwundverschluss 1, 7. In vitro-Anwendung eines elektrischen Gleichstromfeldes an kultivierte Zellen ahmt die physiologischen elektrischen Feld und induziert Richtungszellmigration oder Galvanotaxis. Galvanotaxis in Fibroblasten 8, Fisch Keratinozyten 9, humanen epithelialen und Hornhaut Keratinozyten 10-12 untersucht worden, Lymphozyten 13, Neuroblasten 2 und neuronale Vorläuferzellen 14. Wenn zu dem angelegten Feld ausgesetzt sind, die Mehrzahl der untersuchten Zellen wandern gerichtet in Richtung der kathodenseitigen Pol (-). Dennoch verschiedenen Krebszellen, einschließlich hochmetastatischmenschliche Brustkrebszellen, und der menschliche Prostatakrebs-Zelllinie PC-3M, zu bewegen, um die anodenseitige Pol (+) 15, 16. Verschiedene Mechanismen sind vorgeschlagen Galvanotaxis vermitteln oder die Fähigkeit der Zellen, um das elektrische Feld zu erfassen, einschließlich der Aktivierung erklären der EGF-Rezeptoren 12, die epithelialen Natriumkanals 17, PI3K und PTEN 18, und die Freisetzung von Calciumionen 15, 19. Der Mechanismus ist noch nicht vollständig verstanden, und es ist möglich, dass mehrere Signalwege in Galvanotaxis beteiligt. Die 2-dimensionalen Galvanotaxis Verfahren wir hier zeigen, ist nützlich, um die gerichtete Migration von adhärenten, bewegliche Zellen zu charakterisieren, auf einzelne Zellmigration 10, 12, 17 oder Migration einer Lage von zusammenhängenden Zellen 18, 20. Diese Technik ist aus modifizierten überwachen Peng und Jaffe 21 und Nishimura et al. 10 mit maßgeschneiderten, klarem PVC Kammern mit herausnehmbaren coverslips erlaubt eine einfache Zellentnahme nach Galvanotaxis für die Sekundäranalyse, wie zum Beispiel Immunfluoreszenz-Bildgebung. Die Glasoberfläche der Galvanotaxis Kammern optischen kompatibel, was die Filmbildung bei hoher Vergrößerung und mit fluoreszenzmarkierten Zellen erlaubt. Es ermöglicht auch das experimentelle Design mit Modifizierung der Glasoberfläche, wie die Änderung der Oberflächenbeschichtung oder Ladungen. Spacer aus No. 1 Abdeckglas gebildet in den Kammern verwendet, um den Stromfluss in den Zellen zu minimieren; damit die Joulesche Erwärmung, die proportional zu dem Quadrat des Stromflusses ist, würden die Zellen nicht während des Experimentes hitzen. Die Verbindungs ​​Agar Brücken verhindern einen direkten Kontakt der Elektroden mit den Zellen und Änderung des Mediums pH oder Ionenkonzentration während Galvanotaxis verhindern. Zwei nicht-tumorigene menschliche Prostata-Zelllinien wurden hinsichtlich ihrer Galvanotaxis Reaktion in dieser Studie untersucht. Die PRNs-1-1 22 und 23 sind beide PNT2 SV40 immortalisierte, Wachstumsfaktor-abhängigen Zelllinien, welche die epithelialen Marker Cytokeratin 5, 8, 18 und 19 mit geringer oder keiner Expression des Prostata-spezifisches Antigen (PSA). Beide Zelllinien halten die polygonale Morphologie von normalen Epithelzellen, aber Chromosomenanomalie in Karyotypisierung 22, 24 festgestellt. Obwohl PRN-1-1 und PNT2 teilen ähnliche Verhaltensweisen in den meisten Experimenten zeigen sie Unterschiede bei der Bildung der Struktur und in azinösen Galvanotaxis. In einem 3-D-Matrix, Matrigel, die PRN-1-1 Zellen bilden Hohl acinar Strukturen mit Lumen ähnlich der normalen Prostatagewebe 25. Allerdings sind die PNT2 Zellen bilden feste Kügelchen ohne Lumen oder polarisierten Epithel 26. Die PRNs-1-1-Zellen eine höhere galvanotactic Antwort als die PNT2 in der aktuellen Studie zeigen auch. Die Korrelation zwischen der Bildung von acinar Struktur und Galvanotaxis in PRNs-1-1 schlägt vor, dass die galvanotactic Signale können eine Rolle bei der Organisation der pr spielenostate Drüsengewebe Bewegungen als Reaktion auf endogene elektrische Felder, und stellt weitere Merkmale, um zwischen diesen 2 Zelllinien unterscheiden.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

Die physiologische elektrische Feld dient spezifische biologische Funktionen, wie zB die Leitung der Zellmigration in der Embryonalentwicklung, neuronale Auswachsen und epithelialen Wundheilung. Anlegen eines Gleichstromfeldes, um kultivierte Zellen in vitro induziert gerichteten Zellmigration oder Galvanotaxis. Die 2-dimensionalen Galvanotaxis Verfahren wir hier zeigen, wird mit maßgeschneiderten Poly (vinylchlorid) (PVC) Kammern, Glasoberfläche, Platin-Elektroden und der Verwendung eines motorisierten Tisch, auf dem die Zellen abgebildet werden modifiziert. Die PVC-Kammern und Platinelektroden eine geringe Zytotoxizität und sind erschwinglich und wiederverwendbare. Die Glasoberfläche und der motorisierten Mikroskoptisch zur Verbesserung der Qualität der Bilder und lassen mögliche Änderungen an der Glasoberfläche und Anwendungen zu den Zellen. Wir filmten die Galvanotaxis von zwei nicht-tumor, SV40-immortalisierten Prostata-Zelllinien, PRNs-1-1 und PNT2. Diese beiden Zelllinien zeigen ähnliche Migrationsgeschwindigkeiten und beide wandern zudie Kathode, aber sie zeigen ein unterschiedliches Maß an Richtwirkung in Galvanotaxis. Die über dieses Protokoll erhaltenen Ergebnisse deuten darauf hin, dass die PRN-1-1 und die PNT2 Zelllinien können unterschiedliche intrinsische Eigenschaften, die ihren Richtungsmigrations Antworten zu regieren.

Zwei Linien der Prostatazellen (PRNs-1-1 und PNT2) wurden mit dieser Methode untersucht. Zellen in beiden Leitungen wandern bei ähnlichen Geschwindigkeiten von 1,0 +/- 0,3 um / min im Verlauf von 2-Stunden (5A). , Die Direktionalität zu dem elektrischen Feld ist jedoch 0,7 +/- 0,3 für das PRN-1-1 Linie und 0,2 +/- 0,8 für die PNT2 Leitung (5B). Die Ergebnisse zeigen einen signifikanten Unterschied in der Galvanotaxis dieser beiden Zelllinien (p &lt;0,01, 100-Zellen wurden verfolgt),...

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Verwendung Maßgeschneiderte Galvanotaxis Chambers zu Directional Migration Studie von Prostatazellen

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

utilizing-custom-designed-galvanotaxis-chambers-to-study-directional

Research

JoVE Journal

Biology

9.1K Aufrufe.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Serie: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-gesteuerte gerichtete Wanderung von neuralen Vorläuferzellen in 2D-und 3D-Umgebungen

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Forschung

Biotechnik

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Evaluation of Cancer Stem Cell Migration Mit Abschottung Mikrofluidiksysteme und Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medizin

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Zugverhalten von Zellen in ganglionären Eminenz Kulturen Generated

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Neurowissenschaften

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Live Cell Imaging-Assays Studie Glioblastom Gehirn Tumor Stammzellen Migration und Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Krebsforschung

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analyse der Trunk Neural Crest Cell Migration mit Hilfe eines modifizierten Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Studium der Zellmigration in Microfabricated Kanäle

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

A Galvanotaxis Assay für die Analyse von neuralen Vorläuferzellen Cell Migration Kinetics in einem von außen angelegten Direct Current Electric Field

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Gradient Diffusionssystem, um Chemotaxis in einem 3D-Kollagen-Matrix Untersuchen

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Messung zellulärer Chemotaxis mit ECIS / Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Biologie

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Imaging G Protein-gekoppelten Rezeptor-vermittelte Chemotaxis und seine Signalereignisse in Neutrophilen-like HL60-Zellen

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...